DNA is enzymatically synthesized from deoxyribonucleoside phosphates; attempts to utilize nucleoside phosphates with halogen or sulfur atoms on the purine or pyrimidine moiety as substrates for DNA synthesis in living cells, however, demonstrated that these compounds do not readily penetrate the cell membrane. A cytotoxic nucleotide analog that can enter a living cell intact would be useful as an antimetabolite and potential antitumor agent. The methylphosphonate group was substituted for the phosphate group because the latter may retard membrane penetration. An analog of dTMP, ammonium 5-bromo-2'- deoxyuridine-5' -methylphosphonate (BdUR-MeP) was prepared from 3' -O- acteyl-5-bromodeoxyuridine. Our experiments showed that BdUR-MeP penetrates the cell membrane intact and may be a substrate of TMP kinase. Furthermore, it appears likely that BdUR-MeP may be incorporated intact into the DNA. This hypothesis is based on the total cytotoxicity at 10 micron M BdUR-MeP, the competition with thymidine, the irreversibility of the toxicity, and the light sensitivity of partially inhibited cells. Experiments to provide a direct demonstration of the incorporation of the methylphosphonate nucleotide analog in the DNA of living cells are proposed. The methyl hydrogen atoms of methylphosphonic acid will be exchanged for tritium, and the labeled compound will be esterified at the 5' -position of BdUR-MeP. Chinese hamster cells, human bone marrow cells, and human chronic myeloid leukemic cells will be grown in bottle cultures for 9 days with the 5' -tritium labeled BdUR-MeP, and the medium will be separated from the cells. The cell membrane will be broken with a homogenizer and the extent of tritium labeling in the medium, cell cytoplasm, protein fraction, RNA fraction, and DNA fraction will be determined by suitable fractionation and scintillation techniques. These experiments should provide further information on the mechanism of the cytotoxic action of BdUR-MeP.